Instrumenting scientific ideas
WORLD
PRECISION
INSTRUMENTS
NO Microsensors
ISO-NOPF100, ISO-NOPF200, ISO-NOPF-200-L10, ISO-NOP70L,
ISO-NOP3005, ISO-NOP3020 ISO-NOP30-L, ISO-NOP007, ISO-NOPNM
Serial No._____________________
www.wpiinc.com
INSTRUCTION MANUAL
051316
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Copyright © 2016 by World Precision Instruments, Inc. All rights reserved. No part of this publication
may be reproduced or translated into any language, in any form, without prior written permission of
World Precision Instruments, Inc.
CONTENTS
ABOUT THIS MANUAL ................................................................................................................... 1
INTRODUCTION .............................................................................................................................. 1
Notes and Warnings ................................................................................................................. 2
INSTRUMENT DESCRIPTION ........................................................................................................ 3
Parts List ...................................................................................................................................... 3
Unpacking ................................................................................................................................... 3
OPERATING INSTRUCTIONS ......................................................................................................... 4
Environmental Inuences ....................................................................................................... 4
Temperature ......................................................................................................................... 4
Electrical Interference ...................................................................................................... 4
Attaching the Sensor to the Microsensor Handle ............................................................ 4
Polarizing the Sensor ............................................................................................................... 5
Calibrating the Sensor ............................................................................................................ 6
Understanding SNAP ............................................................................................................... 6
Method 1: SNAP using CuCl .............................................................................................. 6
Method 2: SNAP using CuCl2 ....................................................................................................... 8
Method 3: NO Standards Prepared with NO Gas ....................................................... 9
INSTRUMENT MAINTENANCE .................................................................................................... 12
Storing the Sensor .................................................................................................................. 12
Cleaning the Sensor ...............................................................................................................12
ACCESSORIES.................................................................................................................................12
TROUBLESHOOTING ...................................................................................................................13
SPECIFICATIONS ............................................................................................................................14
APPENDIX A: PREPARING STOCK SOLUTION .........................................................................15
100mM Standard SNAP Solution ........................................................................................15
APPENDIX B: CONSTRUCTING A CALIBRATION CURVE .......................................................16
Predicting the Level of Detectable NO CuCl2 ...........................................................................16
Example for Creating a Calibration Curve .........................................................................17
INDEX ............................................................................................................................................... 21
WARRANTY .....................................................................................................................................23
Claims and Returns ................................................................................................................23
Repairs ....................................................................................................................................... 23
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ABOUT THIS MANUAL
The following symbols are used in this guide:
This symbol indicates a CAUTION. Cautions warn against actions that can
cause damage to equipment. Please read these carefully.
This symbol indicates a WARNING. Warnings alert you to actions that can
cause personal injury or pose a physical threat. Please read these carefully.
NOTES and TIPS contain helpful information.
INTRODUCTION
The NO Microsensors are available in a variety of tip diameters and lengths
for a range of applications. These microsensors incorporate WPI’s proprietary
combination electrode technology whereby the composite graphite nitric oxide
sensing element and separate reference electrode are encased within a single
sensor design.
• Used for in vivo applications, the ISO-NOPF sensors are available in 100µm (WPI
#ISO-NOPF100) and 200µm (WPI #ISO-NOPF200) diameters. They were created
to be exible and are the most durable of all the WPI’s nitric oxide sensors. The
ISO-NOPF is also available with a hypodermic sheath (WPI #ISO-NOPF100H
or ISO-NOPF200H). These sensors are sold in 1-5mm lengths of whole 1mm
increments (1mm, 2mm, 3mm, 4mm, 5mm).
• For use with a tissue bath or in monolayer-style cell culture studies, the ISO-
NOPF200 is available in an L-shape (#ISO-NOPF200-L10), and it is 10mm long.
The L-shaped version is also available in 70mm (WPI #ISO-NOP-70-L) or 30mm
(WPI #ISO-NOP30-L) diameters, both 3mm long.
• When working with microvessels, three options are available. The ISO-NOP007
is 7mm in diameter and 2mm long. The ISO-NOP3005 is 30mm in diameter and
0.5mm long. And, the ISO-NOP3020 is 30mm in diameter and 2mm long.
• For single cell applications, the conical tip of the ISO--NOPNM is only 100mm and
the sensor tip is 150mm long.
Fig. 1 (on the next page) shows a labeled drawing of the carbon ber microsensors.
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2 mm
A or B
Carbon fiber with hydrophobic
gas permeable coating
Hydrophobic
gas permeable
coating
Stainless steel handle
Shielded cable
to ISO-NO meter
A = 2 mm tip length
B = 0.5 mm tip length
Flexible
sensor body
Diameter:
7µm,
30µm,
or
100nm
Diameter:
100µm
or
200µm
Fig. 1—ISO-NOPF microsensor with cable
Notes and Warnings
The NO carbon ber microsensors are robust, but not indestructible. Exercise
caution when handling the NO sensor to avoid actions that could damage the tip.
Do not bring the tip into contact with hard surfaces like stir bars. See ”Unpacking”
on page 3.
CAUTION: DO NOT EXPOSE SENSOR TO ORGANIC SOLVENTS.
CAUTION: Carefully read the “Probe Unpacking” instructions (found in the
sealed sensor case) before handling the sensor.
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INSTRUMENT DESCRIPTION
Parts List
After unpacking, verify that there is no visible damage to the sensor. Verify that all
items are included:
(2 or 3) NO microsensors (quantity depends on the type of sensor)
(2 or 3) Sensor Performance Evaluations (each sensor is tested individually at WPI)
(1) Instruction Manual
Unpacking
Upon receipt of these sensors, make a thorough inspection of the contents
and check for possible damage. Missing cartons or obvious damage to cartons
should be noted on the delivery receipt before signing. Concealed damage
should be reported at once to the carrier and an inspection requested. Please
read the section entitled “Claims and Returns” on page 23 of this manual.
Please contact WPI Customer Service if any parts are missing at 941-371-1003 or
The microsensors are shipped in a sealed, rigid plastic, hinged box with foam
padding to protect them from damage during shipment.
To open the package, carefully cut the seals on either side of the sensor box. The
sensors are packaged in foam holders so that their tips are physically isolated from
contact with the container. To remove a microsensor from the package, use the
thumb and index nger of one hand to separate the slit in the foam which contains
the sensor and use the thumb and index nger of the other hand to grasp the
sensor at its midsection and gently remove it from the container.
KEEP THE SENSOR STORAGE BOX and the documentation in a safe place. The test
date and serial number of each sensor is printed on the bottom of its box. Use of
the sensor should begin within 30 days of receipt.
Returns: Do not return any goods to WPI without obtaining prior approval (RMA
# required) and instructions from WPI’s Returns Department. Goods returned
(unauthorized) by collect freight may be refused. If a return shipment is necessary,
use the original container, if possible. If the original container is not available, use
a suitable substitute that is rigid and of adequate size. For further details, please
read the section entitled “Claims and Returns” on page 23 of this manual.
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OPERATING INSTRUCTIONS
Environmental Inuences
There are two environmental parameters to which NO sensors are quite sensitive:
temperature and electrical interference.
Temperature
The background current (and to a lesser degree) the selectivity of the NO sensor
is aected by temperature. This is due to the eects of temperature on the partial
pressure of dissolved NO gas in liquid samples, on the permeability of the coatings
and on the conductivities of various sensor components. It is recommended that
a calibration procedure be performed at the same temperature as the experiment
and that temperature be held constant during NO measurement.
Electrical Interference
External, electrical noise sources (like magnetic stirrers, uorescent lights, MRI
machines, electric motors, computers, pumps and other electrical instruments)
may couple into the sensor signal path electromagnetically and impose
undesirable signals in the output record. The magnitude of this external noise
depends on the environment of the laboratory. If the interference introduced
by the electrical signals in the environment is large, identify the noise source and
remove it. It is also important to ground and shield the system properly.
TIP: Refer to your free radical analyzer manual for proper grounding and shielding
techniques. (In the TBR4100 or Apollo1000 manuals, see “Grounding and Noise
Concerns” in the Operating Instructions section.)
Attaching the Sensor to the Microsensor Handle
Once removed from the package, plug the microsensor into a microsensor cable
(WPI #91580) connected to the free radical analyzer (Fig. 2).* Be very careful that
the sensor tip does not come into contact with anything which could damage
it. The sensor should plug in easily. If you encounter resistance, it is probably
due to misalignment of the sensor plug with the socket connector inside the
microsensor cable. Simply realign the sensor by gently rotating it until it snaps into
place.
*NOTE: Current WPI free radical analyzers include the TBR4100, TBR1025 and
Apollo1000. Apollo4000 is the original 4-channel WPI free radical analyzer.
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Insert sensor connector into microsensor cable
Microsensor Cable (91580)
Microsensor Cable (91580)
connector
Fig. 2—NO microsensors may be changed or replaced quickly and easily
Polarizing the Sensor
When a non-polarized microsensor is initially connected to a free radical analyzer,
it may display a high (sometimes o-scale) background current. The polarization
voltage applied by the instrument causes a reduction of the background current
to a stabilized baseline value over time. Set the poise voltage to 865mV. (For the
TBR4100/1025, set the Probe Select dial to NO. ) The amount of time required to
reach a stable baseline current varies for each sensor. New sensors typically take
longer, on the order of several hours.
The Performance Evaluation included with your sensor shows the baseline
current and the sensitivity of your sensor when it was quality tested at WPI.
(In addition, it shows the polarization time of your sensor in the WPI labs.) The
baseline value attainable in your lab may be slightly higher or lower, depending
on the temperature* and composition of the test media. For initial performance
verication of an ISO-NOPF in your lab, WPI recommends using the CuCl2
calibration method (Method 2) described on page 8. Once a stable baseline
current is achieved (usually between 500-8000pA), the microsensor is ready for
use.
*NOTE: The background current of the sensor will usually increase with increasing
temperature of the experiment. Although the sensitivity of the sensor does
not change signicantly within the range 20-37°C, it is recommended that the
calibration procedure be performed at the same temperature as the experiment.
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Calibrating the Sensor
Accurate measurements of NO require an accurate calibration. Three calibration
methods are described in this section.
• Method 1 is based on the decomposition of the S-nitrosothiol NO-donor (SNAP)
using CuCl. The NO liberated from SNAP is used to calibrate the sensor.
• Method 2 is similar to Method 1 using CuCl2 as a catalyst. This convenient
method is the one used in WPI laboratories. Experimentally it has been shown
that CuCl2 is less ecient as a catalyst in the conversion of SNAP to NO. (The
conversion ratio is reduced to approximately 60%.) However, it is technically
easier to accomplish than Method 1, and it provides reliable data.
• Method 3 involves preparing aqueous solutions of NO from saturated NO
solutions prepared with NO gas.
WARNING: THIS METHOD USES NO GAS WHICH CAN BE FATAL IF IT IS
MISHANDLED.
Understanding SNAP
SNAP is a stable NO-containing compound that can be used for quantitative
generation of NO in solution. SNAP decomposes to NO and a disulde byproduct
when dissolved in water. However, the rate of decomposition is very slow. The kinetics
of decomposition for this reagent is a function of several parameters including pH,
presence of a catalyst, temperature and light.
In the procedures described here, SNAP is used in combination with a catalyst,
cuprous (I) chloride (CuCl) or cupric (II) chloride (CuCl2), to generate a known
quantity of NO in solution. Note that this protocol does not investigate the eects
of all parameters involved in SNAP decomposition, nor does it propose a model
by which NO is decomposed. The presented procedures provide an empirical
estimation of the amount of generated NO based on the molarity of a standard
(stock) solution of SNAP under a controlled set of parameters.
NOTE: Remember that most NO probes are sensitive to temperature changes. It
is therefore recommended that the calibration of a NO sensor is performed at the
experimental temperature.
Method 1: SNAP using CuCl
This method of calibration results in the 100% conversion of SNAP to NO. The
amount of NO produced, therefore, is based on the nal concentration of SNAP.
NOTE: The described calibration procedure requires the use of cuprous chloride,
CuCl, where CuCl is the active catalyst for the conversion of SNAP to NO. The
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calibration curve assumes only the presence of CuCl and hence a 100% conversion
eciency of SNAP to NO. However, in the presence of oxygen CuCl is readily
oxidized to CuCl2. This will happen naturally if the compound is exposed to air and/
or there is inadequate storage of CuCl. The oxidation product CuCl2 is much less
ecient at catalyzing the conversion of SNAP to NO, and this would appear during
calibration as an apparent low sensitivity of the electrode to NO. Since CuCl is
readily oxidized to CuCl2 special precautions must be taken to keep it in its reduced
state prior to any calibration. It is recommended that CuCl be stored under inert
conditions, and (if used in solution) the solution must be degassed with inert gas
and absent of all oxygen.
NOTE: If your laboratory is not adequately equipped to satisfy the conditions for
storage and use of CuCl, use the CuCl2 method (Method 2) on page 8.
Preparing Solutions
• Solution #1 (Saturated solution of CuCl): Add 150mg CuCl to 500mL distilled
deoxygenated water. The saturated CuCl solution will have a concentration of
approximately 2.4mM at room temperature and should be kept in the dark prior
to use.
NOTE: The distilled water can be deoxygenated by purging with pure nitrogen or
argon gas for 15 minutes.
• Solution #2 (Standard SNAP solution): Add 5.0mg EDTA to 250mL of water and
deoxygenate the solution. Then add 5.6mg of SNAP and dissolve it completely.
TIP: For complete instructions on making standard 100mM SNAP and calculating
the molarity of SNAP solution, see Appendix A, page 15.
Calibration Procedure
1. Within a nitrogen or argon environment, place 10.0mL of Solution #1 (CuCl) in a
20mL vial.
2. Drop a small stirring bar into the solution, and place the vial on a magnetic stirring
plate.
3. Immerse an NO probe into this solution. While stirring allow the sensor to
polarize until the background current stabilizes. The appropriate time for
stabilization and expected baseline current values depend on the model of the
sensor. (See “Specications” on page 14.)
4. As soon as the background current stabilizes, begin recording the current output.
5. Sequentially inject ve aliquots containing 2µL, 4µL, 8mL, 16mL and 32µL of
Solution #2 (SNAP standard solution) into the vial containing Solution #1 (CuCl).
Immediately following the rst addition of SNAP into Solution#1, the current
(pA) output will increase rapidly. Within a few seconds the response will reach
a plateau. Inject the second aliquot (4µL) as soon as the rst signal reaches a
plateau. Add the third aliquot (8mL) as the second signal reaches its plateau. If
aliquots are not added promptly when reaching the previous plateau, the signal
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will slowly decline because generated NO is quickly oxidized to nitrite and nitrate
which will not be detected by the probe. Add the fourth and fth aliquots in the
same manner.
6. Construct a calibration curve by plotting the signal output (pA) against the
concentration (nM) of SNAP. (See “Appendix B: Constructing a Calibration Curve”
on page 16.)
Method 2: SNAP using CuCl2
Preparing Solutions
• Solution #1(CuCl2 solution): 250mL 0.1M CuCl2 in distilled water
• Solution #2 (Standard SNAP solution): Add 5.0mg EDTA to 250mL of water. Then,
add 5.6mg of SNAP and dissolve it completely.
TIP: For complete instructions on making standard 100mM SNAP and calculating
the molarity of SNAP solution, see Appendix A, page 15.
Calibration Procedure
1. Place 20.0mL of Solution #1(CuCl2) in a 20mL vial.
2. Drop a small stirring bar into the solution, and place the vial on a magnetic stirring
plate.
3. Immerse an NO probe into this solution. While stirring allow the background
current to stabilize. The appropriate time for stabilization depends on the model
of the sensor. (See “Specications” on page 14.)
4. As soon as the background current stabilizes, begin recording the current output.
5. Sequentially inject ve aliquots containing 2µL, 4µL, 8mL, 16mL and 32µL of the
SNAP standard solution (Solution #2) into the vial containing Solution #1 (CuCl2).
Immediately following the rst addition of SNAP into Solution#1, the current
(pA) output will increase rapidly. Within a few seconds the response will reach
a plateau. Inject the second aliquot (4µL) as soon as the rst signal reaches a
plateau. Add the third aliquot (8mL) as the second signal reaches its plateau. If
aliquots are not added promptly when reaching the previous plateau, the signal
will slowly decline because generated NO is quickly oxidized to nitrite and nitrate
which will not be detected by the probe. Add the fourth and fth aliquots in the
same manner.
TIP: You can adjust the volume of injected aliquots according to the concentration
of SNAP stock solution. Decrease the volume of aliquot if the electrode is very
sensitive or increase the volume of aliquot if the electrode is less sensitive.
6. Construct a calibration curve by plotting the signal output (pA) against the
concentration (nM) of SNAP. (See “Appendix B: Constructing a Calibration Curve”
on page 16.)
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Method 3: NO Standards Prepared with NO Gas
This method can be used with all NO sensors and has the advantage of allowing
you to calibrate NO sensors in the same environment in which the experimental
measurements will be made. However, it has the disadvantages of added cost,
inconvenience, and greater hazard to the user. All of these factors must be taken
into consideration.
WARNING: NITRIC OXIDE MUST BE HANDLED ONLY IN A WELL-
VENTILATED AREA, TYPICALLY A LABORATORY FUME HOOD WITH
FORCED VENTILATION. THE U.S. OCCUPATIONAL SAFETY AND HEALTH
ADMINISTRATION HAS SET A TIME-WEIGHTED AVERAGE MAXIMUM NO
VALUE AS 25PPM. THAT IS TO SAY, 25PPM IS CITED AS THE MAXIMUM
CONCENTRATION TO WHICH WORKERS MAY BE CONTINUALLY EXPOSED.
BRIEF INHALATION OF CONCENTRATIONS AS LOW AS 200PPM COULD
PRODUCE DELAYED PULMONARY EDEMA WHICH MAY BE FATAL AFTER AN
ASYMPTOMATIC PERIOD OF UP TO 48 HOURS AFTER THE INITIAL EXPOSURE. IT
IS THEREFORE CRITICAL THAT THE PERSONNEL HANDLING THE GAS BE
THOROUGHLY FAMILIAR WITH THE MATERIAL SAFETY DATA SHEET (MSDS) AND
PROPER HANDLING PROCEDURES. THE PRECAUTIONS RECOMMENDED BY THE
GAS MANUFACTURER MUST BE FOLLOWED.
Fig. 3—Setup for preparing a saturated NO aqueous solution must be in a fume hood
with forced ventilation. Nitric oxide is highly toxic, and it escapes into the atmosphere
during prepation of the standards. NO Lecture bottle (14.2L, 98.5%) can be obtained
from Aldrich.
1. Be certain the fume hood is functioning. Inhalation of NO gas is
potentially fatal. See the WARNING above.
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2. Make sure that all ttings and connections are secure. The tubing to be used
should not be permeable to NO. We recommend Tygon® tubing if a polymer
tubing is to be used. This is permeable to NO, but it has the best performance
compared to other polymer tubing of which we are currently aware. Ideally glass
tubing should be used. If Tygon® tubing is used, note that prolonged exposure
to NO aects its properties. Therefore, it is recommended that the tubing be
inspected frequently and that it be replaced when it appears to be brittle. The
pressure regulator and tee purge adaptor should be stainless steel since nitric
oxide is corrosive.
3. Prepare 100mL of a 10% (by weight) KOH solution and place it in the sidearm
ask (Fig. 3). The ask should be sealed with a stopper through which the tubing
passes by means of a Luer tting to a syringe needle which extends almost to
the bottom of the ask. Tubing is used to connect the side arm of the ask to the
vial containing the water to be equilibrated with NO. The KOH solution is used to
remove other nitrogen oxides from the NO gas.
4. Place 20mL of distilled (preferably deionized) water in a small glass vial. Seal the
vial with a stopper and insert (through the stopper) a long syringe needle which
extends almost to the base of the vial. Connect this syringe needle to the tubing
from the KOH ask, as illustrated. Insert an additional shorter syringe needle
which should not extend into the solution. This acts as a pressure relief
during purging.
5. Place the distilled water vial in an ice-water bath. Reducing the temperature
increases the solubility of NO in solution. Thus, when the solution is used at room
temperature, you will be assured of a saturated NO solution.
6. Purge the system with argon (or nitrogen) gas for a period of 30 minutes at a
moderate ow rate such that the pressure is maintained at a safe level (1-2PSI).
When purging, it should be observed that gas is indeed bubbling through the
KOH solution, as well as the distilled water. After 30 minutes turn o the argon
source and switch the tee purge valve to the correct position for purging with NO
from the lecture bottle.
7. If you are using a pure source, purge the system with NO for 5-10 minutes. (If
the source is not pure, it will take longer.) Again, verify that gas is bubbling in the
solution.
WARNING: NO IS NOW ESCAPING FROM THE PRESSURE RELIEF NEEDLE
IN THE STOPPER OF THE DISTILLED WATER VIAL. IT IS IMPERATIVE
THAT THE FUME HOOD BE RUNNING AT MAXIMUM CAPACITY WITH
THE FRONT PANEL CLOSED (IN THE DOWN POSITION).
8. After the time in step 7 has elapsed, turn o the NO source.
9. Immediately remove the two needles from the distilled water vial.
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10. Set the tee purge valve for purging with argon (or nitrogen) gas, and turn on the
argon source. Purge the system for 5-10 minutes at a moderate ow rate. Gas
should be bubbling through the KOH and then escaping from the ask into the
atmosphere. Again, be sure that the fume hood is ventilating well.
11. Turn o the argon (or nitrogen) source, and allow the fume hood to continue to
ventilate for 10-15 minutes to ensure that all traces of NO gas are removed from
the atmosphere.
12. The solution of distilled water should now be saturated with NO. The
concentration of NO produced by this saturation is dependent upon the
temperature. At 0°C, the concentration is approximately 3.3mM, and at 20°C the
concentration is approximately 1.91mM.
13. Dilutions of known concentration can be prepared from this saturated solution.
In preparing a dilution, be careful not to unseal the vial, for this exposes the
solution to atmospheric oxygen.
Once the dilutions are prepared, it is a simple matter to calibrate the instrument.
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INSTRUMENT MAINTENANCE
Storing the Sensor
STANDBY: When not being used for a short period of time (such as overnight),
the microsensor should remain attached to the microsensor cable and kept dry
(not immersed in solution). Before the next experiment, immerse the sensor
in the experimental solution (like, Kreb’s Buer). The background current will
increase until reaching a stable value. Do not be alarmed if the background current
becomes elevated. This is associated with the hydration of the sensor and will not
negatively aect the sensor’s performance.
LONG-TERM: If the microsensor will not be used for more than three or four days,
then it may be stored dry by removing it from the microsensor cable and returning
it to the case in which it was shipped, being very careful to avoid making contact
with the sensor tip.
The NO microsensor is a maintenance-free consumable sensor. When its
performance is no longer satisfactory, remove it from the microsensor cable and
dispose of it, replacing it with a new one.
Cleaning the Sensor
The sensor should be cleaned after each use by suspending the tip in distilled
water for 20-30 minutes to dissolve salts and remove particles which may have
accumulated on the membrane. If the sensor was used in a protein-rich solution,
the tip should rst be soaked in a protease solution for several minutes to remove
protein build-up and then rinsed with distilled water. Enzymatic detergent (Enzol,
WPI #7363-4) can also be used. The sensor can be sterilized chemically using an
appropriate disinfectant (Cidex, WPI #7364). If necessary, gently dab the sensor
with a Kimwipes® to remove residue.
NOTE: ALWAYS rinse with distilled water before storing the sensor dry.
ACCESSORIES
Part Number Description
7363-4 Enzol Enzymatic Detergent, 1 gallon
7364 Cidex Disinfecting Solution
91580 Microsensor Cable
47510 ProGuide Postion/Holder with Base
47520 ProGuide Position/Holder
47530 ProGuide Plus Position/Holder with ne
adjustment
47540 ProGuide Plus with Base
SNAP25 SNAP, 25mg vial
SNAP50 SNAP, 50mg vial
SNAP100 SNAP, 100mg vial
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TROUBLESHOOTING
Issue Possible Cause Solution
Baseline current is below
specied range.
The poise voltage (sensor
setting) may be incorrectly set.
Set the poise voltage to 865mV. (For the
TBR, choosing the NO sensor setting
selects 865mV automatically.) Set the
range at 10nA.
The sensor may be nearing the
end of its usable life.
Perform a 5-point calibration set using
the standard. If the sensor responds
linearly within the desired concentration
range, it is still useable. See “Calibrating
the Sensor” on page 6.
Unstable
baseline
If the baseline hasn’t stabilized
after 2 hours, the polarizing
solution may be contaminated.
Prepare fresh polarizing solution. Use
0.1M CuCl2 only.
External electrical interferences
may be the problem.
Identify and isolate electrical
interferences.
Calibration
dataset is
not linear.
Uneven aliquots may have been
used.
Check the pipetter calibration.
The stock solutions have
deteriorated.
Prepare fresh standard solution. See
“Calibrating the Sensor” on page 6.
Sensitivity
below range
specied
Foreign materials have been
adsorbed on the sensor’s
surface.
If the foreign materials are proteins, use
and enzymatic cleanser like Enzol (WPI
#7363-4) to remove the contaminant.
The sensor has reached end of
its usable life.
Replace the sensor.
NOTE: If you have a problem/issue with your NO Microsensor that falls outside
the denitions of this troubleshooting section, rst perform the CuCl2 Calibration
Procedure (Method 2) exactly as describe on page 8 of this manual and contact
the WPI Technical Support team at 941-371-1003 or [email protected].
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SPECIFICATIONS
The NO Microsensors conform to the following specications:
NOPF200 NOPF200-L10 NOPF100 NOP70-L NOP3005 NOP3020 NOP30-L NOP007 NOPNM
Outside Diameter (µm) 200 200 100 70 30 30 30 7 Conical
tip:100
Available Length
2
(mm) 1-5
1
10 1-5
1
3 0.5 2 3 2 150mm
Response Time (sec.) < 5 < 5 < 5 < 3 < 3 < 3 < 3 < 3 < 3
Lowest Detection
Limit/Range (nM)
0.2 0.2 0.2 1 1 1 1 0.5 0.5
Nominal Sensitivity-New
Sensor
2
(pA/nM)
≥20 ≥50 ≥10 ≥10 ≥1≥1.5 ≥1≥1≥0.5
Baseline Drift none none none none none none none none none
Poise Voltage (mV) 865 865 865 865 865 865 865 865 865
Typical Quiescent Base-
line Current, 25ºC (pA)
2500 3500 2000 3500 1000 500 500 300 300
Acceptable Baseline
Range (pA)
500-
8000
500-8000 500-
8000
2000-
6000
150-
3500
500-
5000
20-
6500
200-
1500
50-100
Polarization Time (hrs) 2+ 8+ 2+ 2+ 1+ 1+ 1+ 1+ 1+
1Sensor length varies in 1mm increments (for example, 1mm, 2mm, 3mm...).
2Sensor length is proportional to sensitivity.
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APPENDIX A: PREPARING STOCK SOLUTION
100mM Standard SNAP Solution
SNAP is a green crystalline compound that is sold in 25mg, 50mg and 100mg
vials (WPI # SNAP25, SNAP50, SNAP100). Both the crystalline form and the liquid
solution of SNAP are photo-sensitive and tend to degrade over time. Wrap the
vial of SNAP compound in aluminum foil and store it in the freezer to slow its
degradation. Similarly, store the bottle of SNAP solution in an amber bottle or wrap
it with aluminum foil and store it in the refrigerator.
NOTE: The decomposition of SNAP at low temperature, in the dark and in the
absence of trace metal ions proceeds slowly because of the EDTA (a chelating
reagent).
WPI technicians recommend making fresh standard SNAP solution daily to ensure
accurate calibration of NO sensors. Concentration of SNAP decreases to 5-10% of
its nominal value after approximately 4-5 hours.
To make a 100mM solution of SNAP:
1. Accurately weigh out 5.0mg EDTA (a preservative) and place it in a clean, dry
bottle that will hold at least 250mL.
2. Use a clean, dry 250mL volumetric ask to accurately measure 250mL of HPLC
pure water (HPLC grade, Sigma).
TIP: If your research demands an oxygen-free sample, you can de-oxygenate this
solution by purging it with pure nitrogen or argon gas for 15 minutes.
3. Pour the water into the bottle with EDTA. Replace the cap and shake it for a few
seconds to dissolve the EDTA. It dissolves rapidly.
4. Accurately weight out 5.6mg of crushed, crystalline SNAP.
TIP: Crush any clumps of SNAP powder with a clean instrument like a glass
stirring rod, a popsicle stick or a tooth pick. If you prefer, place the 5.6mg SNAP
on a small piece of lter paper, fold the paper in half and rub it gently between
your ngers to break up any clumps. Be careful not to spill any of the compound.
5. Add the 5.6mg SNAP to the EDTA solution. Verify that none of the green SNAP
compound is left on your lter paper or measuring tray. Replace the cap and
shake it for a few seconds until the green ecks dissolve into solution.
6. Store this standard solution in an amber bottle, if available, or alternatively, wrap
aluminum foil around the bottle to limit light intrusion. This solution should be
refrigerated.
16 World Precision Instruments
The concentration of SNAP (f.w.= 220.3) in the stock solution is calculated as
follows:
[C] = [A•W/(M•V)]1000
[C] = concentration of SNAP (µM)
A = purity of SNAP
W = weight of SNAP (mg)
M = formula weight of SNAP (220.3g/mol)
V = volume of the solution (L)
If SNAP purity is 98.5%, the concentration of standard SNAP stock solution describe
above is:
[C] = [0.985 x 5.6mg/(220.3g/mol x 0.25L)] x 1000 = 100.1µM
NOTE: The purity of SNAP used is extremely important to ensure an accurate
calibration. We recommend the use of high grade SNAP with minimal purity of 98%
or better.
APPENDIX B: CONSTRUCTING A CALIBRATION
CURVE
Because NO sensors can be calibrated in a linear fashion, the magnitude of every
signal should almost double as the volume of SNAP solution added is doubled in
the course of the calibration. The response should be linear from 10 to 1000nM.
Use the recorded data to construct a calibration curve by plotting the signal
output (for example, in pA) against the concentration of SNAP added at that
time. Note that every addition of SNAP solution corresponds with a particular
NO concentration. The sensitivity of the NO probe can be established from the
gradient or slope of the response curve. After the sensitivity of the NO probe is
established, software (like Data-Trax) can be programmed to display data in either
concentration directly (nM or mM) or redox current (pA or nA).
Predicting the Level of Detectable NO CuCl2
Experiments have shown that SNAP is decomposed instantaneously under the
following set of experimental conditions:
• Temperature 25°C
• Catalyst solution 0.1M CuCl2
• SNAP (WPI, 98% purity) - Fresh standard SNAP solution
• CuCl2 is at equilibrium with ambient air (aerobic conditions)
SNAP (RSNO) decomposes to NO and a disulde byproduct according to the
following equation:
2 RSNO➡2NO + RS – SR
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